The mechanocoupling of energy stored in triphosphate nucleosides provide the power necessary for many in vivo processes. One major class of enzymes, the AAA family, couples ATP hydrolysis to mechanical motions essential in a variety of cellular pathways including (but not limited to) protein degradation, organelle maintenance, replication and recombination. The protein p97 (also known as valosin-containing protein) is one of the most widely studied members of this family and has therefore become a representative member from which general principles of AAA proteins may be inferred. First discovered in 1990, p97 is highly abundant in the cell (composing nearly 1% of the cytosol), hexamerizes, and forms a stacked-ring shaped complex in solution. p97 is also believed to play a key role in the degradation pathway of IkBa, which results in the down-regulation of apoptosis in cancer cells and explains the observation of increased p97 presence in numerous cancer lines. Structurally, each monomer is composed of two hydrolysis domains (D1 and D2 with only D2 being catalytically active under standard cellular conditions), an N-terminal domain that interacts with effector proteins, a C-terminal domain, and linker regions between them. In this proposal we suggest computational experiments to expand our understanding of the structure and function of p97 in addition to developing small molecule inhibitors that target its active site. Initial in silico work will focus on the conformational structures and motions inherent to the major hydrolysis states through the use of long-times scale molecular dynamics (MD) simulations. In addition, discrepancies between low and high resolution experimental structures will be addressed. In an attempt to discover small molecules that inhibit p97, virtual screening will then be performed against structures resulting from the MD simulations using docking methods in conjunction with the relaxed complex scheme. Top candidate molecules will then be experimentally tested by our collaborators and their results may then be used in guiding further screening calculations. Molecules identified as top inhibitors will then be refined through lead optimization, which will be assisted through the development of a novel lead optimization methodology. Finally, hydrolysis pathways will be analyzed through free energy calculations with combined quantum mechanical/molecular mechanics calculations to further our understanding of residues, water molecules, and ions in the active site. Results of simulations will advance our understanding of p97 structure and function on multiple time and length scales while also developing new small molecule inhibitors that target this highly important protein. Additionally, the lead optimization methods developed herein will allow for increased accuracy at a reduced cost in structure based drug design. PUBLIC HEALTH RELEVANCE: Results from simulations proposed here might potentially have a significant impact on public health through advancing our understanding of the structure, function, and hydrolysis mechanism of the highly abundant enzyme p97. The importance to various cellular pathways makes p97 an interesting chemotherapeutic target, and one project described here aims to discover high-affinity inhibitors of this target (of which there are currently none) and could potentially lead to drug development targeting cancer cells. Additionally, the development of lead optimizations methods could result in improved structure based drug design methods for a variety of enzyme targets.